Shane M. Hanlon

Unlikely Remedy: Fungicide Clears Infection from Pathogenic Fungus in Larval Southern Leopard Frogs (Lithobates sphenocephalus)

Amphibians are often exposed to a wide variety of perturbations. Two of these, pesticides and pathogens, are linked to declines in both amphibian health and population viability. Many studies have examined the separate effects of such perturbations; however, few have examined the effects of simultaneous exposure of both to amphibians. In this study, we exposed larval southern leopard frog tadpoles (Lithobates sphenocephalus) to the chytrid fungus Batrachochytrium dendrobatidis and the fungicide thiophanate-methyl (TM) at 0.6 mg/L under laboratory conditions. The experiment was continued until all larvae completed metamorphosis or died. Overall, TM facilitated increases in tadpole mass and length. Additionally, individuals exposed to both TM and Bd were heavier and larger, compared to all other treatments. TM also cleared Bd in infected larvae. We conclude that TM affects larval anurans to facilitate growth and development while clearing Bd infection. Our findings highlight the need for more research into multiple perturbations, specifically pesticides and disease, to further promote amphibian heath.

Development of antimicrobial peptide defenses of southern leopard frogs, Rana sphenocephala, against the pathogenic chytrid fungus, Batrachochytrium dendrobatidis

Amphibian species face the growing threat of extinction due to the emerging fungal pathogen Batrachochytrium dendrobatidis, which causes the disease chytridiomycosis. Antimicrobial peptides (AMPs) produced in granular glands of the skin are an important defense against this pathogen. Little is known about the ontogeny of AMP production or the impact of AMPs on potentially beneficial symbiotic skin bacteria. We show here that Rana (Lithobates) sphenocephala produces a mixture of four AMPs with activity against B. dendrobatidis, and we report the minimum inhibitory concentration (MIC) of synthesized replicates of these four AMPs tested against B. dendrobatidis. Using mass spectrometry and protein quantification assays, we observed that R. sphenocephala does not secrete a mature suite of AMPs until approximately 12 weeks post-metamorphosis, and geographically disparate populations produce a different suite of peptides. Use of norepinephrine to induce maximal secretion significantly reduced levels of culturable skin bacteria.

The Impact of Pesticides on the Pathogen Batrachochytrium dendrobatidis Independent of Potential Hosts

Amphibians around the world are experiencing the greatest organismal decline in recent history. Xenobiotics, such as pesticides, and pathogenic biotic perturbations, including the fungus Batrachochytrium dendrobatidis (Bd), have played major roles in amphibian decreases. We conducted laboratory culture studies to determine the effects of three pesticides {carbaryl, glyphosate, and thiophanate-methyl [TM; Topsin-M(R) (Cerexagri-Nisso LLC)]} on Bd zoospore production and zoosporangia growth. We applied Bd to pesticides mixed in an agar culture to simulate pathogen introduction to a system with pre-existing pesticides (Bd addition). Alternatively, pesticides were applied to pre-established Bd to simulate pesticide introduction after Bd establishment (pesticide addition). We then measured Bd zoosporangia and zoospore production. All pesticides significantly inhibited zoospore production; however, glyphosate and TM were more effective at doing so than carbaryl. In addition, only carbaryl and glyphosate inhibited zoosporangia production. Our data suggest that carbaryl and glyphosate are equally effective at inhibiting both zoosporangia and zoospore production; however, TM is selectively toxic to zoospores but not zoosporangia. One possible explanation for this observation could be that TM is toxic to zoospores but not the protective zoosporangia. In the case of pesticides applied to established Bd cultures, all pesticides caused significant mortality in both zoosporangia and zoospores, and no differences were found among pesticides. We conclude that examining pesticide and pathogen interactions independent of hosts provides mechanistic understanding of such interactions before and after host infection or contamination.

Optimal digestion theory does not predict the effect of pathogens on intestinal plasticity

One prediction of optimal digestion theory is that organisms will increase the relative length of their digestive tracts when food resources become limited. We used theory of optimal digestion to test whether tadpoles can adjust the relative length of their intestines when challenged with the fungal pathogen Batrachochytrium dendrobatidis (Bd). The degree of tadpole mouthpart damage, a symptom of Bd infections that reduces food consumption, was associated positively with the length of tadpole intestines relative to their body size, consistent with optimal digestion theory. After controlling for mouthpart damage, tadpoles exposed to Bd had shorter intestines relative to their body size, opposite to the predictions of optimal digestion theory. One explanation of why tadpoles with higher Bd loads have shorter relative intestinal lengths is that they divert energy from maintaining intestinal and overall growth towards anti-parasite defences.

Batrachochytrium dendrobatidis exposure effects on foraging efficiencies and body size in anuran tadpoles.

Chytridiomycosis, the amphibian disease caused by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), is fatal to adults of many species. Bd is largely sublethal to amphibian larvae; however, it is known to reduce larval (i.e. tadpole) growth rates, with possible long-term effects on population dynamics and fitness. We conducted an experiment to test how Bd altered southern leopard frog Lithobates sphenocephalus tadpole mouthpart damage, percentage of food ingested, and subsequent body size. We examined our results using path analyses. We hypothesized that Bd would increase mouthpart damage, causing less food to be ingested, and ultimately reduce body size. In our model, both Bd exposure and increased mouthpart damage significantly reduced food ingested and subsequent body size. However, our study provides evidence against the long-standing hypothesis of mouthpart damage as a pathway for Bd-induced reductions in larval group. Here we provide evidence for reduced foraging efficiency (percentage of food ingested) as a mechanism for Bd-induced reductions in body size. This work highlights the importance of studying the sublethal effects of Bd on larval amphibians.

Mouthparts of Southern Leopard Frog, Lithobates sphenocephalus , Tadpoles not Affected by Exposure to a Formulation of Glyphosate

This study evaluated the impacts of Roundup® on tadpole mouthpart damage as a mechanism for reduced growth and developmental rates in Lithobates sphenocephalus (Southern leopard frog) tadpoles. We did not find evidence that Roundup® damages larval mouthparts, nor was there a significant relationship between mouthpart damage and either body condition or developmental rate. However, the highest concentration of Roundup® significantly stunted development compared to all other treatments. Although we observed a significant effect of Roundup® on developmental rate, we conclude that mouthpart damage is likely not a mechanism for this life history response.

Detection of amphibian chytrid fungus on waterfowl integument in natural settings

The chytrid fungus Batrachochytrium dendrobatidis (Bd), the causal agent of the amphibian disease chytridiomycosis, has spread at an alarming rate since its discovery. Bd was initially thought to only infect keratinizing epithelial cells in amphibians, a core component of amphibian skin. However, recent studies have detected Bd on the integument of non-amphibian hosts. We conducted a survey of 3 duck species (gadwalls, green-winged teals, and mallards) to determine whether Bd DNA could be found on their feet. Bd was found on the feet, by quantitative PCR, of individuals from all 3 species (5/11 gadwalls, 4/8 green-winged teals, and 13/21 mallards), though there were no significant differences in zoospore presence or load between species. We conclude that these waterfowl species may act as vector hosts for Bd, adding to the growing list of potential waterfowl vectors. Future studies are needed to determine whether Bd on waterfowl feet is viable and infectious to amphibian hosts.